Mass Spectrometry and Affinity Chromatography Techniques for Studying Arsenic-Binding Proteins in Human Cells

Arsenic is one of the most important environmental agents in causing chronic human disease. Elevated levels of arsenic in drinking water may affect >100 million people around the world. A wide variety of adverse health effects, most seriously, cancers of bladder, lung, urinary tract, and skin, have been attributed to chronic exposure to arsenic. However, the biochemical mechanisms responsible for these effects caused by arsenic remain unclear, but may be mediated by the binding of trivalent arsenicals to thiol groups in proteins, thereby changing the conformation of these proteins and inhibiting their functions. If some of the affected proteins are responsible for cellular repair of DNA damage, for example, the inhibition of these proteins could lead to carcinogenesis.
To study interaction of arsenic with proteins, we have developed an affinity selection technique, coupled with mass spectrometry, to select and identify specific arsenic-binding proteins from a large pool of cellular proteins. Controlled experiments using proteins either containing free cysteine(s) or inactive cysteine showed that the arsenic affinity column specifically captured the proteins containing free cysteine(s) available to bind to arsenic. The technique was able to capture and identify trace amounts of bovine biliverdin reductase B present as a minor impurity in the commercial preparation of carbonic anhydrase II, demonstrating the ability to identify arsenic-binding proteins in the presence of a large excess of non-specific proteins. Application of the technique to the analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction, and 24 proteins in the membrane/organelle fraction that could bind to arsenic. This added substantially to the current list of only a few known arsenic-binding proteins.
A number of arsenic-binding proteins identified using the affinity chromatography tandem mass spectrometry approach were of particular interest because of their important biological functions. For example, DNA-dependent protein kinase, ATP-dependent helicase II (Ku70), and topoisomerase 2 alpha, are involved in DNA repair and maintaining genome stability. Several other proteins modulate the redox status of cells, e.g. peroxiredoxin-1 and thioredoxin, and apoptosis, e.g., lamin A and heat shock cognate protein. This work shows that arsenic can bind to these proteins in cell extracts. How arsenic affects the function of these proteins in biological systems will have to be confirmed by studying arsenic interaction with proteins in living cells.